Methods and compositions for treating metastatic breast cancer and other cancers in the brain

ABSTRACT

A composition comprising at least one AAV vector formulated for central nervous system delivery is described. The composition comprises at least one expression cassette which contains sequences encoding an anti-neoplastic immunoglobulin construct for delivery to the brain operably linked to expression control sequences therefor and a pharmaceutically acceptable carrier. The anti-neoplastic immunoglobulin construct may be an immunoglobulin modified to have decreased or no measurable affinity for neonatal Fc receptor (FcRn). Also provided are methods of using these constructs in preparing pharmaceutical compositions and uses thereof in anti-neoplastic regimens, particularly for primary and/or metastatic cancers of the brain.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED IN ELECTRONIC FORM

Applicant hereby incorporates by reference the Sequence Listing materialfiled in electronic form herewith. This file is labeled“14-7028PCT_ST25.txt”.

BACKGROUND OF THE INVENTION

Brain metastases are a common and devastating sequelae of breast cancerfor which treatment options are few and inadequate. 6-16% of breastcancer patients develop central nervous system (CNS) metastases. Thesepatients have a 20% one-year and 1.3% five-year median survival from thetime of diagnosis. DiStefano A, et al, Cancer. 1979; 44:1913-1918;Takakura K, et al, Metastatic tumors of the central nervous system.Tokyo: Igaku-Shoin, 1982; Hall W A, et al, Long-term survival withmetastatic cancer to the brain. Med Oncol. 2000 November; 17(4):279-86;Pieńkowski T, Zielinski C C. Trastuzumab treatment in patients withbreast cancer and metastatic CNS disease. Ann Oncol. 2010 May;21(5):917-24. Surgical resection of brain metastases is ofteninfeasible, and chemotherapeutic agents are mostly excluded from the CNSby the blood brain barrier (BBB) [Nakayama A, et al, Antitumor Activityof TAK-285, an Investigational, Non-Pgp Substrate HER2/EGFR KinaseInhibitor, in Cultured Tumor Cells, Mouse and Rat Xenograft Tumors, andin an HER2-Positive Brain Metastasis Model, J Cancer. 2013 Aug. 16;4(7)]. Alternative therapies to treat breast cancer brain metastases areneeded.

Breast cancers that overexpress the HER2 receptor tyrosine kinase have ahigh propensity to metastasize to the CNS and comprise 25-30% of allbreast cancer cases [Bendell J C, et al, Central nervous systemmetastases in women who receive trastuzumab-based therapy for metastaticbreast carcinoma. Cancer. 2003 Jun. 15; 97(12):2972-7]. Trastuzumab(Herceptin®) is a first-line therapeutic immunoglobulin G (IgG)monoclonal antibody (mAb) directed toward HER2; this antibody has beenreported to significantly improve survival of patients with HER2positive disease [Lin N U, et al, Brain metastases: the HER2 paradigm.Clin Cancer Res. 2007 Mar. 15; 13(6):1648-55; Palmieri D, et al, Her-2overexpression increases the metastatic outgrowth of breast cancer cellsin the brain. Cancer Res. 2007 May 1; 67(9):4190-8]. However, patientsthat benefit from trastuzumab often experience simultaneous progressionof CNS disease because mAbs do not cross the BBB [Nakayama, citedabove]. Injecting trastuzumab directly into the CNS has been proven tobe safe, and intrathecal administration of trastuzumab to patients withleptomeningeal carcinomatosis has been reported to increase overallsurvival from 2 to 13.5 months [Zagouri F, et al, Intrathecaladministration of trastuzumab for the treatment of meningealcarcinomatosis in HER2-positive metastatic breast cancer: a systematicreview and pooled analysis. Breast Cancer Res Treat. 2013 May;139(1):13-22]. Leptomeningeal carcinomatosis is associated with animpaired, rather than an intact, blood-brain barrier. Park, E-J, et al,J Controlled Release, 163 (2012), 277-284 report that focused ultrasoundbursts combined with circulating microbubbles can temporarilypermeabilize both the blood brain barrier and the blood tumor barrierfor trastuzumab.

While current therapies have led to an improved control of the systemicdisease, treatment of metastatic dissemination of human breast cancerinto the CNS is a great therapeutic challenge.

SUMMARY OF THE INVENTION

An anti-neoplastic composition is provided which comprises at least oneAAV vector formulated for delivery to the central nervous system,wherein said composition comprises at least one expression cassettewhich contains sequences encoding an anti-neoplastic immunoglobulinproduct for delivery to the CNS operably linked to expression controlsequences therefor and a pharmaceutically acceptable carrier. In oneexample, the anti-neoplastic immunoglobulin construct comprises animmunoglobulin modified to have decreased or no measurable affinity forneonatal Fc receptor (FcRn). Suitably, this composition is effective foruse in retarding the growth of a tumor in the brain and/or for reducingtumor size and/or for increasing progression-free survival of thesubject.

In one aspect, a composition as provided herein comprises an AAV viralvector having an AAV9 capsid and having packaged therein an expressioncassette encoding an anti-Her2 IgG antibody or a functional fragmentthereof which comprises an anti-Her2 heavy chain which has disruptedbinding for FcRn.

In another aspect, a method for retarding the growth of a tumor in thebrain is provided, which involves administering a composition asdescribed herein to the central nervous system e.g., intrathecally. Inone aspect, the composition is administered in the absence of chemicalor physical disruption of the blood brain barrier.

In yet another aspect, the invention provides a method for treating aneoplasm in the brain by administering a composition as described hereinto a subject in need thereof.

In yet another aspect, the invention provides an anti-neoplastic regimencomprising administering a composition as described herein incombination with an antibody or other biologic drug, a small moleculeanti-neoplastic agent, radiation, and/or a chemotherapeutic agent.

Still other aspects and advantages of the invention will be readilyapparent from the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the amino acid sequence of the heavy chain of thepolypeptide of trastuzumab, with the sequence listing numbering [SEQ IDNO:25] above the sequence and the conventional Eu (IMGT) numbering belowthe sequence.

FIG. 2 provides a survival curve of mice given 1×10¹¹ GC ICV ofAAV9.trastuzumab or AAV9.201IA prophylactically, then implanted withBT474-M1.ffluc tumor cells in the brain 21 days after vectoradministration. The median survival of 201IA group (sham treatment) isshown to be 66 days, whereas the median survival of the group treatedwith AAV9.trastuzumab is 99 days, a 33% increase in survival rate.

DETAILED DESCRIPTION OF THE INVENTION

The compositions and regimens described herein are useful for deliveryof anti-neoplastic immunoglobulin constructs to the central nervoussystem. Compositions described herein comprising AAV-Ig are well suitedfor central nervous system (CNS) cancers (neoplasms), and particularlyfor those located in the brain.

As used herein, the term “CNS neoplasms” includes primary or metastaticcancers, which may be located in the brain (intracranial), meninges(connective tissue layer covering brain and spinal cord), or spinalcord. Examples of primary CNS cancers could be gliomas (which mayinclude glioblastoma (also known as glioblastoma multiforme),astrocytomas, oligodendrogliomas, and ependymomas, and mixed gliomas),meningiomas, medulloblastomas, neuromas, and primary CNS lymphoma (inthe brain, spinal cord, or meninges), among others. Examples ofmetastatic cancers include those originating in another tissue or organ,e.g., breast, lung, lymphoma, leukemia, melanoma (skin cancer), colon,kidney, prostate, or other types that metastasize to brain.

As used herein, an “anti-neoplastic” immunoglobulin construct (includingantibody or antibody fragment as defined herein) encodes apolypeptide-based moiety which binds to a cell-surface antigen orreceptor located on a cancer cell or solid tumor and which inhibits orprevents the growth and spread of tumors, or malignant cells in anon-solid tumor, and optionally, reduces the size of tumors. Theanti-neoplastic immunoglobulin polypeptides can function by a number ofmechanisms, e.g., inhibiting tumor cell growth by blocking a growthfactor receptor, cross-linking cell membrane antigens to deliver signalsthat control the cell cycle, blocking angiogenesis, blocking DNA repairpost chemotherapy, or even inducing cell death. Alternatively, they caninfluence tumor growth indirectly by activating host immune effectorfunctions such as antibody-dependent and complement-mediated cellcytotoxicity. In one embodiment, the anti-neoplastic effect of thecompositions and regimens described herein can be measured by reductionof tumor size and/or by an increased progression-free survival rate ascompared to subjects which are untreated or treated with other regimens.

The term “immunoglobulin” is used herein to include antibodies,functional fragments thereof, and immunoadhesins. Antibodies may existin a variety of forms including, for example, polyclonal antibodies,monoclonal antibodies, camelized single domain antibodies, intracellularantibodies (“intrabodies”), recombinant antibodies, multispecificantibody, antibody fragments, such as, Fv, Fab, F(ab)₂, F(ab)₃, Fab′,Fab′-SH, F(ab′)₂, single chain variable fragment antibodies (scFv),tandem/bis-scFv, Fc, pFc′, scFvFc (or scFv-Fc), disulfide Fv (dsfv),bispecific antibodies (bc-scFv) such as BiTE antibodies; camelidantibodies, resurfaced antibodies, humanized antibodies, fully humanantibodies, single-domain antibody (sdAb, also known as NANOBODY®),chimeric antibodies, chimeric antibodies comprising at least one humanconstant region, and the like. “Antibody fragment” refers to at least aportion of the variable region of the immunoglobulin that binds to itstarget, e.g., the tumor cell.

The term “heterologous” when used with reference to a protein or anucleic acid indicates that the protein or the nucleic acid comprisestwo or more sequences or subsequences which are not found in the samerelationship to each other in nature. For instance, the nucleic acid istypically recombinantly produced, having two or more sequences fromunrelated genes arranged to make a new functional nucleic acid. Forexample, in one embodiment, the nucleic acid has a promoter from onegene arranged to direct the expression of a coding sequence from adifferent gene. Thus, with reference to the coding sequence, thepromoter is heterologous.

As used herein, an “expression cassette” refers to a nucleic acidmolecule which comprises an immunoglobulin gene(s) (e.g., animmunoglobulin variable region, an immunoglobulin constant region, afull-length light chain, a full-length heavy chain or another fragmentof an immunoglobulin construct), promoter, and may include otherregulatory sequences therefor, which cassette may be delivered via agenetic element (e.g., a plasmid) to a packaging host cell and packagedinto the capsid of a viral vector (e.g., a viral particle). Typically,such an expression cassette for generating a viral vector contains theimmunoglobulin sequences described herein flanked by packaging signalsof the viral genome and other expression control sequences such as thosedescribed herein.

As described above, the term “about” when used to modify a numericalvalue means a variation of ±10%, unless otherwise specified.

As used throughout this specification and the claims, the terms“comprise” and “contain” and its variants including, “comprises”,“comprising”, “contains” and “containing”, among other variants, isinclusive of other components, elements, integers, steps and the like.The term “consists of” or “consisting of” are exclusive of othercomponents, elements, integers, steps and the like.

For expression from an AAV vector, the amino acid sequences for ananti-neoplastic immunoglobulin construct are selected from those whichhave been published, those which are commercially available, and thecoding sequences described herein. Anti-neoplastic immunoglobulins asdescribed herein may target a human epidermal growth factor receptor(HER), such as HER2. An example of trastuzumab is a recombinant IgG1kappa, humanized monoclonal antibody that selectively binds with highaffinity in a cell-based assay (Kd=5 nM) to the extracellular domain ofthe human epidermal growth factor receptor protein. The commerciallyavailable product is produced in CHO cell culture. See, e.g.,http://www.drugbank.ca/drugs/DB00072. The amino acid sequences of thetrastuzumab light chains 1 and 2 and heavy chains 1 and 2, as well assequences obtained from a study of the x-ray structure of trastuzumab,are provided on this database at accession number DB00072, whichsequences are incorporated herein by reference. See, also,212-Pb-TCMC-trastuzumab [Areva Med, Bethesda, Md.]. Another antibody ofinterest includes, e.g., pertuzumab, a recombinant humanized monoclonalantibody that targets the extracellular dimerization domain (SubdomainII) of the human epidermal growth factor receptor 2 protein (HER2). Itconsists of two heavy chains and two lights chains that have 448 and 214residues respectively. FDA approved Jun. 8, 2012. The amino acidsequences of its heavy chain and light chain are provided, e.g., inwww.drugbank.ca/drugs/DB06366 (synonyms include 2C4, MOAB 2C4,monoclonal antibody 2C4, and rhuMAb-2C4) on this database at accessionnumber DB06366. In addition to HER2, other HER targets may be selected.

For example, MM-121/SAR256212 is a fully human monoclonal antibody thattargets the HER3 receptor [Merrimack's Network Biology] and which hasbeen reported to be useful in the treatment of non-small cell lungcancer (NSCLC), breast cancer and ovarian cancer. SAR256212 is aninvestigational fully human monoclonal antibody that targets the HER3(ErbB3) receptor [Sanofi Oncology]. Another anti-Her3/EGFR antibody isRG7597 [Genentech], described as being useful in head and neck cancers.Another antibody, margetuximab (or MGAH22), a next-generation,Fc-optimized monoclonal antibody (mAb) that targets HER [MacroGenics],may also be utilized.

Alternatively, other human epithelial cell surface markers and/or othertumor receptors or antigens may be targeted. Examples of other cellsurface marker targets include, e.g., 5T4, CA-125, CEA (e.g., targetedby labetuzumab), CD3, CD19, CD20 (e.g., targeted by rituximab), CD22(e.g., targeted by epratuzumab or veltuzumab), CD30, CD33, CD40, CD44,CD51 (also integin α_(v)β₃), CD133 (e.g., glioblastoma cells), CTLA-4(e.g., Ipilimumab used in treatment of, e.g., neuroblastoma), Chemokine(C-X-C Motif) Receptor 2 (CXCR2) (expressed in different regions inbrain; e.g., Anti-CXCR2 (extracellular) antibody #ACR-012 (AlomeneLabs)); EpCAM, fibroblast activation protein (FAP) [see, e.g., WO2012020006 A2, brain cancers], folate receptor alpha (e.g., pediatricependymal brain tumors, head and neck cancers), fibroblast growth factorreceptor 1 (FGFR1) (see, et al, WO2012125124A1 for discussion treatmentof cancers with anti-FGFR1 antibodies), FGFR2 (see, e.g., antibodiesdescribed in WO2013076186A and WO2011143318A2), FGFR3 (see, e.g.,antibodies described in U.S. Pat. No. 8,187,601 and WO2010111367A1),FGFR4 (see, e.g., anti-FGFR4 antibodies described in WO2012138975A1),hepatocyte growth factor (HGF) (see, e.g., antibodies inWO2010119991A3), integrin α₅β₁, IGF-1 receptor, gangioloside GD2 (see,e.g., antibodies described in WO2011160119A2), ganglioside GD3,transmembrane glycoprotein NMB (GPNMB) (associated with gliomas, amongothers and target of the antibody glembatumumab (CR011), mucin, MUC1,phosphatidylserine (e.g., targeted by bavituximab, PeregrinePharmaceuticals, Inc], prostatic carcinoma cells, PD-L1 (e.g., nivolumab(BMS-936558, MDX-1106, ONO-4538), a fully human gG4, e.g., metastaticmelanoma], platelet-derived growth factor receptor, alpha (PDGFR α) orCD140, tumor associated glycoprotein 72 (TAG-72), tenascin C, tumornecrosis factor (TNF) receptor (TRAIL-R2), vascular endothelial growthfactor (VEGF)-A (e.g., targeted by bevacizumab) and VEGFR2 (e.g.,targeted by ramucirumab). Other antibodies and their targets include,e.g., APN301 (hu14.19-IL2), a monoclonal antibody [malignant melanomaand neuroblastoma in children, Apeiron Biologics, Vienna, Austria]. See,also, e.g., monoclonal antibody, 8H9, which has been described as beinguseful for the treatment of solid tumors, including metastatic braincancer. The monoclonal antibody 8H9 is a mouse IgG1 antibody withspecificity for the B7H3 antigen [United Therapeutics Corporation]. Thismouse antibody can be humanized. Still other immunoglobulin constructstargeting the B7-H3 and/or the B7-H4 antigen may be used in theinvention. Another antibody is S58 (anti-GD2, neuroblastoma). Cotara™[Perregrince Pharmaceuticals] is a monoclonal antibody described fortreatment of recurrent glioblastoma. Other antibodies may include, e.g.,avastin, ficlatuzumab, medi-575, and olaratumab. Still otherimmunoglobulin constructs or monoclonal antibodies may be selected foruse in the invention. See, e.g., Medicines in Development Biologics,2013 Report, pp. 1-87, a publication of PhRMA's Communications & PublicAffairs Department. (202) 835-3460, which is incorporated by referenceherein.

Once the target and immunoglobulin are selected, the coding sequencesfor the selected immunoglobulin (e.g., heavy and/or light chain(s)) maybe obtained and/or synthesized. Methods for sequencing a protein,peptide, or polypeptide (e.g., as an immunoglobulin) are known to thoseof skill in the art. Once the sequence of a protein is known, there areweb-based and commercially available computer programs, as well asservice based companies which back translate the amino acids sequencesto nucleic acid coding sequences. See, e.g., backtranseq by EMBOSS,http://www.ebi.ac.uk/Tools/st/; Gene Infinity(http://www.geneinfinity.org/sms/sms_backtranslation.html); ExPasy(http://www.expasy.org/tools/). In one embodiment, the RNA and/or cDNAcoding sequences are designed for optimal expression in human cells.

Codon-optimized coding regions can be designed by various differentmethods. This optimization may be performed using methods which areavailable on-line (e.g., GeneArt), published methods, or a company whichprovides codon optimizing services, e.g., DNA2.0 (Menlo Park, Calif.).One codon optimizing algorithm is described, e.g., in US InternationalPatent Publication No. WO 2015/012924, which is incorporated byreference herein. See also, e.g., US Patent Publication No. 2014/0032186and US Patent Publication No. 2006/0136184. Suitably, the entire lengthof the open reading frame (ORF) for the product is modified. However, insome embodiments, only a fragment of the ORF may be altered. By usingone of these methods, one can apply the frequencies to any givenpolypeptide sequence, and produce a nucleic acid fragment of acodon-optimized coding region which encodes the polypeptide.

A number of options are available for performing the actual changes tothe codons or for synthesizing the codon-optimized coding regionsdesigned as described herein. Such modifications or synthesis can beperformed using standard and routine molecular biological manipulationswell known to those of ordinary skill in the art. In one approach, aseries of complementary oligonucleotide pairs of 80-90 nucleotides eachin length and spanning the length of the desired sequence aresynthesized by standard methods. These oligonucleotide pairs aresynthesized such that upon annealing, they form double strandedfragments of 80-90 base pairs, containing cohesive ends, e.g., eacholigonucleotide in the pair is synthesized to extend 3, 4, 5, 6, 7, 8,9, 10, or more bases beyond the region that is complementary to theother oligonucleotide in the pair. The single-stranded ends of each pairof oligonucleotides are designed to anneal with the single-stranded endof another pair of oligonucleotides. The oligonucleotide pairs areallowed to anneal, and approximately five to six of thesedouble-stranded fragments are then allowed to anneal together via thecohesive single stranded ends, and then they ligated together and clonedinto a standard bacterial cloning vector, for example, a TOPO® vectoravailable from Invitrogen Corporation, Carlsbad, Calif. The construct isthen sequenced by standard methods. Several of these constructsconsisting of 5 to 6 fragments of 80 to 90 base pair fragments ligatedtogether, i.e., fragments of about 500 base pairs, are prepared, suchthat the entire desired sequence is represented in a series of plasmidconstructs. The inserts of these plasmids are then cut with appropriaterestriction enzymes and ligated together to form the final construct.The final construct is then cloned into a standard bacterial cloningvector, and sequenced. Additional methods would be immediately apparentto the skilled artisan. In addition, gene synthesis is readily availablecommercially.

The immunoglobulin genes described herein may be used to express the“wild-type”, a published or commercially available, or other knownconstant immunoglobulin domains or can be engineered to decreaseaffinity for, or ablate, binding to the Fc binding site present onimmunoglobulins. There are several different types of Fc receptors,which are classified based on the type of antibody that they recognize.As used herein, “FcRn” refers to the neonatal Fc receptor that bindsIgG. It is similar in structure to MHC class I protein. In humans, it isencoded by the FCGRT gene. The Fc receptor is located on various cellstypes, including, e.g., the epithelial cells of the blood brain barrier.The term “FcRn binding domain” as used herein refers to a protein domainthat directly or indirectly binds to the FcRn. The FcRn may be amammalian FcRn. In further embodiments, the FcRn is a human FcRn. AnFcRn binding domain binding directly to an FcRn is an antibody Fcregion. Meanwhile, regions capable of binding to a polypeptide such asalbumin or IgG, which has human FcRn-binding activity, can indirectlybind to human FcRn via albumin, IgG, or such. Thus, such a humanFcRn-binding region may be a region that binds to a polypeptide havinghuman FcRn-binding activity. The term “Fc region” as used herein refersto an FcRn-binding domain that directly binds to FcRn, a mammalian FcRn,or a human FcRn. In particular, an Fc region is an Fc region of anantibody. The Fc region may be a mammalian Fc region or moreparticularly a human Fc region. In particular, the Fc region may belocated within the second and third constant domain of a humanimmunoglobulin (CH2 and CH3). Further, the Fc region may be the hinge ofCH2 and CH3. In one embodiment, the immunoglobulin construct is an IgG.In a further embodiment, the Fc region is an Fc region of human IgG1.Other Ig isotypes can be used as well.

Because these binding domains are located within the constant region ofan IgG heavy chain (regions CH2 and CH3), the amino acid positionsprovided herein for modification in trastuzumab can be readilydetermined by preparing an alignment with another immunoglobulin heavychain selected for modification in order to identify the correspondingamino acid number. Methods and computer programs for preparing suchalignments are available and well known to those of skill in the art.The amino acid positions referred to in this application are based uponthe numbering of trastuzumab as provided in SEQ ID NO: 3 and 25 (heavychain) and SEQ ID NO: 4 (light chain). Substitutions may also be writtenas (amino acid identified by single letter code)-position #-(amino acididentified by single letter code) whereby the first amino acid is thesubstituted amino acid and the second amino acid is the substitutingamino acid at the specified position. The terms “substitution” and“substitution of an amino acid” and “amino acid substitution” as usedherein refer to a replacement of an amino acid in an amino acid sequencewith another one, wherein the latter is different from the replacedamino acid. Methods for replacing an amino acid are well known to theskilled in the art and include, but are not limited to, mutations of thenucleotide sequence encoding the amino acid sequence. Methods of makingamino acid substitutions in IgG are described, e.g., for WO 2013/046704,which is incorporated by reference for its discussion of amino acidmodification techniques, although this document describes increasingFcRn affinity, rather than decreasing or ablating binding affinity asdescribed herein.

The term “amino acid substitution” and its synonyms described above areintended to encompass modification of an amino acid sequence byreplacement of an amino acid with another, substituting amino acid. Thesubstitution may be a conservative substitution. The term conservative,in referring to two amino acids, is intended to mean that the aminoacids share a common property recognized by one of skill in the art. Theterm non-conservative, in referring to two amino acids, is intended tomean that the amino acids which have differences in at least oneproperty recognized by one of skill in the art. For example, suchproperties may include amino acids having hydrophobic nonacidic sidechains, amino acids having hydrophobic side chains (which may be furtherdifferentiated as acidic or nonacidic), amino acids having aliphatichydrophobic side chains, amino acids having aromatic hydrophobic sidechains, amino acids with polar neutral side chains, amino acids withelectrically charged side chains, amino acids with electrically chargedacidic side chains, and amino acids with electrically charged basic sidechains. Both naturally occurring and non-naturally occurring amino acidsare known in the art and may be used as substituting amino acids inembodiments. Thus, a conservative amino acid substitution may involvechanging a first amino acid having a hydrophobic side chain with adifferent amino acid having a hydrophobic side chain; whereas anon-conservative amino acid substitution may involve changing a firstamino acid with an acidic hydrophobic side chain with a different aminoacid having a different side chain, e.g., a basic hydrophobic side chainor a hydrophilic side chain. Still other conservative ornon-conservative changes change be determined by one of skill in theart.

In still other embodiments, the substitution at a given position will beto an amino acid, or one of a group of amino acids, that will beapparent to one of skill in the art in order to accomplish an objectiveidentified herein.

In one embodiment, an immunoglobulin construct as defined herein isengineered so that the native sequence located on the conserved regionof the immunoglobulin Fc region is ablated to eliminate binding to theFcRn and to minimize or eliminate transport of the proteinaceousimmunoglobulin constructs across the blood brain barrier (out of the CNSarea) and into the systemic circulation. In one example, this may beaccomplished by altering one or more amino acids of the FcRn-bindingdomain, e.g., by modification of the codon for the selected aminoacid(s).

For example, the immunoglobulin may be modified in one or more of thecodons encoding the amino acid reside at position Y436 (aa459 of SEQ IDNO: 25), S254 (aa277 of SEQ ID NO:25), I253 (aa276 of SEQ ID NO: 25),and/or H435 (aa458 of SEQ ID NO: 25) to another suitable amino acid,e.g., alanine (Ala, A). However, other positions involved infunctionally binding to FcRn may be mutated, such as, e.g., 1250 (aa 273of SEQ ID NO:25), M252 (aa 275 of SEQ ID NO: 25), S254 (aa 277 of SEQ IDNO: 25), 1256 (aa 279 of SEQ ID NO: 25), P257 (aa 280 of SEQ ID NO: 25),P271 (aa 294 of SEQ ID NO:25), T307 (aa 330 of SEQ ID NO:25), Q311 (aa334 of SEQ ID NO: 25), D376 (aa 399 of SEQ ID NO: 25), E380 (aa 403 ofSEQ ID NO: 25), M428 (aa 451 of SEQ ID NO: 25), and/or N434 (aa 457 ofSEQ ID NO: 25), or combinations one or more of these with each other, orwith the other modifications described herein. Other suitablemodifications may be located at I253 (aa 276 of SEQ ID NO: 25), S254 (aa277 of SEQ ID NO: 25), K288 (aa 311 of SEQ ID NO: 25), V305 (aa 328 ofSEQ ID NO: 25), Q311 (aa 334 of SEQ ID NO: 25), D312 (aa 335 of SEQ IDNO: 25), K317 (aa 340 of SEQ ID NO: 340), K360 (aa 383 of SEQ ID NO:25), Q362 (aa 385 of SEQ ID NO: 25), E380 (aa 403 of SEQ ID NO: 25),S415 (aa 438 of SEQ ID NO: 25), S424 (aa 447 of SEQ ID NO: 25), H433 (aa456 of SEQ ID NO: 25), N434 (aa 457 of SEQ ID NO: 25), H435 (aa 458 ofSEQ ID NO: 25), and/or Y436 (aa 459 of SEQ ID NO: 25), or combinationsof two or more. As described above, corresponding locations in other IgGheavy chain CH2 and CH2 may be selected. Reference to “one or more”herein is intended to encompass the individual embodiments of, forexample, 1, 2, 3, 4, 5. In additional embodiments, the term “one ormore” includes a number of substitutions in a polypeptide describedherein that would yield at least about 85% identity, at least 90%identity, at least about 95% identity, or at least about 99% identity tothe trastuzumab heavy chain variable region SEQ ID NO: 3, light chainvariable region SEQ ID NO:4, heavy chain SEQ ID NO: 25 or another aminoacid sequence identified herein.

In addition, mutations that enhance complement-dependent cytotoxicity(CDC) and/or antibody-dependent cell-mediated cytotoxicity (ADCC)functions may be incorporated in the trastuzamab variants describedherein. In further embodiments, such a mutation facilitates the killingof the tumor cells by immune cells. Examples of suitable amino acidmodifications to enhance ADCC function are described in, e.g., US PatentPublication No. 2008/0118501; A Nasume, et al, Drug Des Devel Ther,2009, 3; 7-16, publ online Sep. 21, 2009. G A Lazar et al, Proc NatlAcad Sci, vol. 103, no. 11, p. 4005-4010 (Mar. 14, 2006); and G L Moore,et al, MAbs, 2010 March-April; 2(2): 181-189, among others.

The heavy chain amino acid numbering used herein to identify thelocation of the mutants is based on the EU numbering system [IMGT uniquenumbering, Edelman, G. M. et al., Proc. Natl. Acad. USA, 63, 78-85(1969);http://www.imgtorg/IMGTScientificChart/-Numbering/Hu_IGHGnber.html]] andrefer to positions in an FcRn-binding domain, in particular in an Fcregion. In a similar fashion, substitutions are indicated as for example“EU387R” or “EU440E”, wherein the number given after “EU” indicates theposition of the substitution according the EU numbering, and the letterafter the number is the substituted amino acid given in the one lettercode. Other numbering systems include, e.g., Kabat, E. A., T. T. Wu, H.M. Perry, K. S. Gottesman, C. Foeler. (1991) Sequences of Proteins ofImmunological Interest. No. 91-3242 U. S. Public Health Services,National Institutes of Health, Bethesda).

In one embodiment, an anti-Her2 antibody is selected for a compositionand method as described herein. In one embodiment, the selected antibodyis trastuzumab. The amino acid sequences of trastuzumab have beendescribed, e.g., in P. Carter et al, Proc Natl. Acad Sci., 89:4285-4289(May 1982). The amino acid sequence of the trastuzumab heavy chain isprovided in FIG. 1, showing both the sequence listing [SEQ ID NO: 25]and EU numbering systems. The amino acid sequence of the trastuzumabheavy chain variable region is shown in [SEQ ID NO: 3] and thetrastuzumab light chain variable region is provided in the appendedsequence listing [SEQ ID NO: 4]. In order to express trastuzumab, anovel nucleic acid molecule has been designed which contains codonswhich have been selected for optimal expression of the trastuzumabpolypeptides in humans. Further, the novel nucleic acid moleculeincludes a heterologous leader sequence for each the heavy chain andlight chain of trastuzumab, which encodes the IL-2 signal leader peptidefused upstream of the heavy and chain polypeptides composed of thevariable and constant regions. However, another heterologous leadersequence may be substituted for one or both of the IL-2 signal/leaderpeptide. Signal/leader peptides may be the same or different for eachthe heavy chain and light chain immunoglobulin constructs. These may besignal sequences which are natively found in an immunoglobulin (e.g.,IgG), or may be from a heterologous source. Such heterologous sourcesmay be a cytokine (e.g., IL-2, IL12, IL18, or the like), insulin,albumin, β-glucuronidase, alkaline protease or the fibronectin secretorysignal peptides, amongst others. The promoter(s) can be selected fromdifferent sources, e.g., human cytomegalovirus (CMV) immediate-earlyenhancer/promoter, the SV40 early enhancer/promoter, the JC polymoviruspromoter, myelin basic protein (MBP) or glial fibrillary acidic protein(GFAP) promoters, herpes simplex virus (HSV-1) latency associatedpromoter (LAP), rouse sarcoma virus (RSV) long terminal repeat (LTR)promoter, neuron-specific promoter (NSE), platelet derived growth factor(PDGF) promoter, hSYN, melanin-concentrating hormone (MCH) promoter,CBA, matrix metalloprotein promoter (MPP), and the chicken beta-actinpromoter.

The expression cassette described herein may contain at least oneinternal ribosome binding site, i.e., an IRES, located between thecoding regions of the heavy and light chains. Alternatively the heavyand light chain may be separated by a furin-2a self-cleaving peptidelinker [see, e.g., Radcliffe and Mitrophanous, Gene Therapy (2004), 11,1673-1674. The expression cassette may contain at least one enhancer,i.e., CMV enhancer. Still other enhancer elements may include, e.g., anapolipoprotein enhancer, a zebrafish enhancer, a GFAP enhancer element,and brain specific enhancers such as described in WO 2013/1555222,woodchuck post hepatitis post-transcriptional regulatory element.Additionally, or alternatively, other, e.g., the hybrid humancytomegalovirus (HCMV)-immediate early (IE)-PDGR promoter or otherpromoter-enhancer elements may be selected. To enhance expression theother elements can be introns (like promega intron or chimeric chickenglobin-human immunoglobulin intron).

As provided herein, with respect to the numbering of the engineerednucleic acid molecule in SEQ ID NO:1, a nucleic acid sequence encodingthe heavy chain polypeptide of trastuzumab is characterized by theleader sequence (1-60 of SEQ ID NO:1), nucleic acids 61 to 423 are thecoding region for the immunoglobulin heavy chain (HC) variable sequence,nucleic acids 439 to 714 are the coding region for the HC constantregion 1, nucleic acids 715 to 1410 are the coding region for the HCconstant regions 2 and 3. The IRES is located at nucleic acids 1422-2012of SEQ ID NO: 1 between the trastuzumab heavy chain and the leadersequence of the trastuzumab light chain coding sequence. The variableregion of the trastuzumab light chain variable sequence is atnucleotides 2070-2391 of SEQ ID NO: 1; the light chain constant regionis located at nucleic acids 2407 to 2711 of SEQ ID NO:1.

Also encompassed herein are nucleic acid sequences encoding thetrastuzumab immunoglobulin polypeptides [e.g., the heavy chain, thelight chain, or fragments thereof, which fragments may include, e.g.,complementarity determining regions (CDR) 1, 2 and/or 3, a constantregion (1, 2, or 3) of SEQ ID NO:1, or a sequence which is at leastabout 85% identical thereto, at least about 90%, at least about 95%identical thereto, or at least about 99% identical thereto to SEQ ID NO:1, or a fragment thereof coding for an immunoglobulin polypeptide (e.g.,the heavy chain, the light chain, or fragments thereof [e.g., the heavychain, the light chain, or fragments thereof (e.g., a variable region(including, e.g., complementarity determining regions (CDR) 1, 2 and/or3), a constant region (1, 2, or 3))], which encode polypeptides andfragments thereof having the same amino acid sequence as provided hereinfor the trastuzumab without any FcRN modifications.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region(e.g., any one of the modified ORFs provided herein when compared andaligned for maximum correspondence over a comparison window ordesignated region) as measured using a BLAST or BLAST 2.0 sequencecomparison algorithms with default parameters described below, or bymanual alignment and visual inspection (see, e.g., NCBI web site or thelike). As another example, polynucleotide sequences can be comparedusing Fasta, a program in GCG Version 6.1. Fasta provides alignments andpercent sequence identity of the regions of the best overlap between thequery and search sequences. For instance, percent sequence identitybetween nucleic acid sequences can be determined using Fasta with itsdefault parameters (a word size of 6 and the NOPAM factor for thescoring matrix) as provided in GCG Version 6.1, herein incorporated byreference. Generally, these programs are used at default settings,although one skilled in the art can alter these settings as needed.Alternatively, one of skill in the art can utilize another algorithm orcomputer program that provides at least the level of identity oralignment as that provided by the referenced algorithms and programs.This definition also refers to, or can be applied to, the compliment ofa sequence. The definition also includes sequences that have deletionsand/or additions, as well as those that have substitutions. As describedbelow, the preferred algorithms can account for gaps and the like.Preferably, identity exists over a region that is at least about 25, 50,75, 100, 150, 200 amino acids or nucleotides in length, and oftentimesover a region that is 225, 250, 300, 350, 400, 450, 500 amino acids ornucleotides in length or over the full-length of an amino acid ornucleic acid sequences.

Typically, when an alignment is prepared based upon an amino acidsequence, the alignment contains insertions and deletions which are soidentified with respect to a reference AAV sequence and the numbering ofthe amino acid residues is based upon a reference scale provided for thealignment. However, any given AAV sequence may have fewer amino acidresidues than the reference scale. In the present invention, whendiscussing the parental sequence, the term “the same position” or the“corresponding position” refers to the amino acid located at the sameresidue number in each of the sequences, with respect to the referencescale for the aligned sequences. However, when taken out of thealignment, each of the proteins may have these amino acids located atdifferent residue numbers. Alignments are performed using any of avariety of publicly or commercially available Multiple SequenceAlignment Programs. Sequence alignment programs are available for aminoacid sequences, e.g., the “Clustal X”, “MAP”, “PIMA”, “MSA”,“BLOCKMAKER”, “MEME”, and “Match-Box” programs. Generally, any of theseprograms are used at default settings, although one of skill in the artcan alter these settings as needed. Alternatively, one of skill in theart can utilize another algorithm or computer program which provides atleast the level of identity or alignment as that provided by thereferenced algorithms and programs. See, e.g., J. D. Thomson et al,Nucl. Acids. Res., “A comprehensive comparison of multiple sequencealignments”, 27(13):2682-2690 (1999).

In another embodiment, a modified anti-Her2 antibody having its affinityfor FcRn ablated and retaining effective anti-neoplastic activity isprovided. One or more amino acid modifications may be selected to ablatefunctional binding to FcRn. In one embodiment, the mutation lowers thebinding affinity of the trastuzumab immunoglobulin for FcRn to less than10% of the native protein. Suitably, the immunoglobulins with thesemutations bind substantially normally to all other Fc receptors. Forexample, the immunoglobulin may be modified at least one of positionY436, S254, 1253, and/or H435 to an alanine or other amino acid, orcombinations of one or more of these with each other, or one, two ormore with one or more of the modifications described herein. However,other positions involved in functional binding to FcRn may be mutated,such as, e.g., T250 (aa 273 of SEQ ID NO:25), M252 (aa 275 of SEQ ID NO:25), S254 (aa 278 of SEQ ID NO: 25), T256 (aa 280 of SEQ ID NO: 25),P257 (aa 281 of SEQ ID NO: 25), P271 (aa 294 of SEQ ID NO:25), T307 (aa330 of SEQ ID NO:25), Q311 (aa 334 of SEQ ID NO: 25), D376 (aa 399 ofSEQ ID NO: 25), E380 (aa 403 of SEQ ID NO: 25), M428 (aa 451 of SEQ IDNO: 25), and/or N434 (aa 457 of SEQ ID NO: 25), or combinations one ormore of these with each other, or with the other modifications describedherein. Other suitable modifications may be located at I253 (aa 276 ofSEQ ID NO: 25), S254 (aa 278 of SEQ ID NO: 25), K288 (aa 311 of SEQ IDNO: 25), V305 (aa 328 of SEQ ID NO: 25), Q311 (aa 334 of SEQ ID NO: 25),D312 (aa 335 of SEQ ID NO: 25), K317 (aa 340 of SEQ ID NO: 340), K360(aa 383 of SEQ ID NO: 25), Q362 (aa 385 of SEQ ID NO: 25), E380 (aa 403of SEQ ID NO: 25), S415 (aa 438 of SEQ ID NO: 25), S424 (aa 447 of SEQID NO: 25), H433 (aa 456 of SEQ ID NO: 25), N434 (aa 457 of SEQ ID NO:25), H435 (aa 458 of SEQ ID NO: 25), and/or Y436 (aa 459 of SEQ ID NO:25), or combinations of two or more, e.g., by substitution with anotheramino acid which retains the desired anti-neoplastic activity. Stillother mutations may be incorporated. See, e.g., Kuo and Aveson, mAbs,3:5, 422-430 (September/October 2011) and Shield, J Biol Chem, 2001,276: 659-6604. Once the amino acid sequence is selected, the nucleicacid sequences can be designed and/or the previously described sequencesmay be engineered as described above. These modifications are made byengineering the nucleic acid coding region using site directedmutagenesis or other genetic engineering techniques which are known tothose of skill in the art.

Similar modifications may be engineered into another selected anti-HER2immunoglobulin construct, or alternately, into another anti-neoplasticimmunoglobulin construct as described herein.

In one embodiment, the immunoglobulin genes described herein areengineered into a genetic element (e.g., a plasmid) useful forgenerating AAV vectors which transfer the immunoglobulin constructsequences carried thereon. The selected vector may be delivered to a anAAV packaging cell by any suitable method, including transfection,electroporation, liposome delivery, membrane fusion techniques, highvelocity DNA-coated pellets, viral infection and protoplast fusion.Stable packaging cells can also be made. The methods used to make suchconstructs are known to those with skill in nucleic acid manipulationand include genetic engineering, recombinant engineering, and synthetictechniques. See, e.g., Molecular Cloning: A Laboratory Manual, ed. Greenand Sambrook, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2012).

AAV Vectors

An AAV vector as described herein can comprise one or more nucleic acidsequences, each of which encodes one or more of the heavy and/or lightchain polypeptides, or other polypeptides, of an anti-neoplasticimmunoglobulin construct. Suitably, a composition contains one or moreAAV vectors which contain all of the polypeptides which form ananti-neoplastic construct in vivo. For example, a full-length antibodyconsists of four polypeptides: two identical copies of a heavy (H) chainpolypeptide and two copies of a light (L) chain polypeptide. Each of theheavy chains contains one N-terminal variable (VH) region and threeC-terminal constant (CH1, CH2 and CH3) regions, and each light chaincontains one N-terminal variable (VL) region and one C-terminal constant(CL) region. The variable regions of each pair of light and heavy chainsform the antigen binding site of an antibody. In this respect, an AAVvector as described herein can comprise a single nucleic acid sequencethat encodes the two heavy chain polypeptides (e.g., constant variable)and the two light chain polypeptides of an immunoglobulin construct.Alternatively, the AAV vector can comprise a first expression cassettethat encodes at least one heavy chain constant polypeptides and at leastone heavy chain variable polypeptide, and a second expression cassettesthat encodes both light chain polypeptides of an immunoglobulinconstruct. In yet another embodiment, the AAV vector can comprise afirst expression cassette encoding a first heavy chain polypeptide, asecond expression cassette encoding a second heavy chain polypeptide, athird expression cassette encoding a first light chain polypeptide, anda fourth expression cassette encoding a second light chain polypeptide.

Typically, an expression cassette for an AAV vector comprises an AAV 5′inverted terminal repeat (ITR), the immunoglobulin construct codingsequences and any regulatory sequences, and an AAV 3′ ITR. However,other configurations of these elements may be suitable. A shortenedversion of the 5′ ITR, termed ΔITR, has been described in which theD-sequence and terminal resolution site (trs) are deleted. In otherembodiments, the full-length AAV 5′ and 3′ ITRs are used.

Where a pseudotyped AAV is to be produced, the ITRs in the expressionare selected from a source which differs from the AAV source of thecapsid. For example, AAV2 ITRs may be selected for use with an AAVcapsid having a particular efficiency for targeting CNS or tissues orcells within the CNS. In one embodiment, the ITR sequences from AAV2, orthe deleted version thereof (ΔITR), are used for convenience and toaccelerate regulatory approval. However, ITRs from other AAV sources maybe selected. Where the source of the ITRs is from AAV2 and the AAVcapsid is from another AAV source, the resulting vector may be termedpseudotyped. However, other sources of AAV ITRs may be utilized.

The abbreviation “sc” refers to self-complementary. “Self-complementaryAAV” refers a construct in which a coding region carried by arecombinant AAV nucleic acid sequence has been designed to form anintra-molecular double-stranded DNA template. Upon infection, ratherthan waiting for cell mediated synthesis of the second strand, the twocomplementary halves of scAAV will associate to form one double strandedDNA (dsDNA) unit that is ready for immediate replication andtranscription. See, e.g., D M McCarty et al, “Self-complementaryrecombinant adeno-associated virus (scAAV) vectors promote efficienttransduction independently of DNA synthesis”, Gene Therapy, (August2001), Vol 8, Number 16, Pages 1248-1254. Self-complementary AAVs aredescribed in, e.g., U.S. Pat. Nos. 6,596,535; 7,125,717; and 7,456,683,each of which is incorporated herein by reference in its entirety.

The expression cassette typically contains a promoter sequence as partof the expression control sequences, e.g., located between the selected5′ ITR sequence and the immunoglobulin construct coding sequence. Tissuespecific promoters, constitutive promoters, regulatable promoters [see,e.g., WO 2011/126808 and WO 2013/04943], or a promoter responsive tophysiologic cues may be used may be utilized in the vectors describedherein. In addition to a promoter, an expression cassette and/or avector may contain other appropriate transcription initiation,termination, enhancer sequences, efficient RNA processing signals suchas splicing and polyadenylation (polyA) signals; sequences thatstabilize cytoplasmic mRNA; sequences that enhance translationefficiency (i.e., Kozak consensus sequence); sequences that enhanceprotein stability; and when desired, sequences that enhance secretion ofthe encoded product.

These control sequences are “operably linked” to the immunoglobulinconstruct gene sequences. As used herein, the term “operably linked”refers to both expression control sequences that are contiguous with thegene of interest and expression control sequences that act in trans orat a distance to control the gene of interest.

In one embodiment, a self-complementary AAV is provided. This viralvector may contain a A5′ ITR and an AAV 3′ ITR. In another embodiment, asingle-stranded AAV viral vector is provided. Methods for generating andisolating AAV viral vectors suitable for delivery to a subject are knownin the art. See, e.g., U.S. Pat. No. 7,790,449; U.S. Pat. No. 7,282,199;WO 2003/042397; WO 2005/033321, WO 2006/110689; and U.S. Pat. No.7,588,772 B2]. In one system, a producer cell line is transientlytransfected with a construct that encodes the transgene flanked by ITRsand a construct(s) that encodes rep and cap. In a second system, apackaging cell line that stably supplies rep and cap is transientlytransfected with a construct encoding the transgene flanked by ITRs. Ineach of these systems, AAV virions are produced in response to infectionwith helper adenovirus or herpesvirus, requiring the separation of therAAVs from contaminating virus. More recently, systems have beendeveloped that do not require infection with helper virus to recover theAAV—the required helper functions (i.e., adenovirus E1, E2a, VA, and E4or herpesvirus UL5, UL8, UL52, and UL29, and herpesvirus polymerase) arealso supplied, in trans, by the system. In these newer systems, thehelper functions can be supplied by transient transfection of the cellswith constructs that encode the required helper functions, or the cellscan be engineered to stably contain genes encoding the helper functions,the expression of which can be controlled at the transcriptional orposttranscriptional level. In yet another system, the transgene flankedby ITRs and rep/cap genes are introduced into insect cells by infectionwith baculovirus-based vectors. For reviews on these production systems,see generally, e.g., Zhang et al., 2009, “Adenovirus-adeno-associatedvirus hybrid for large-scale recombinant adeno-associated virusproduction,” Human Gene Therapy 20:922-929, the contents of each ofwhich is incorporated herein by reference in its entirety. Methods ofmaking and using these and other AAV production systems are alsodescribed in the following U.S. patents, the contents of each of whichis incorporated herein by reference in its entirety: U.S. Pat. Nos.5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213; 6,491,907;6,660,514; 6,951,753; 7,094,604; 7,172,893; 7,201,898; 7,229,823; and7,439,065.

The available space for packaging may be conserved by combining morethan one transcription unit into a single expression cassette, thusreducing the amount of required regulatory sequences. For example, asingle promoter may direct expression of a single cDNA or RNA thatencodes two or three or more genes, and translation of the downstreamgenes are driven by IRES sequences. In another example, a singlepromoter may direct expression of a cDNA or RNA that contains, in asingle open reading frame (ORF), two or three or more genes separatedfrom one another by sequences encoding a self-cleavage peptide (e.g.,2A) and/or a protease recognition site (e.g., furin). The ORF thusencodes a single polyprotein, which, either during or after translation,is cleaved into the individual proteins (such as, e.g., heavy chain andlight chain). It should be noted, however, that although these IRES andpolyprotein systems can be used to save AAV packaging space, they canonly be used for expression of components that can be driven by the samepromoter. In another alternative, the transgene capacity of AAV can beincreased by providing AAV ITRs of two genomes that can anneal to formhead to tail concatamers.

In the examples below, an AAV9 vector is described for expressingtrastuzumab directly in the CNS to treat breast cancer CNS metastases.AAV9 vectors are described, e.g., in U.S. Pat. No. 7,906,111, which isincorporated herein by reference. However, other sources of AAV capsidsand other viral elements may be selected, as may other immunoglobulinconstructs and other vector elements. Methods of generating AAV vectorshave been described extensively in the literature and patent documents,including, e.g., WO 2003/042397; WO 2005/033321, WO 2006/110689; U.S.Pat. No. 7,588,772 B2. The source of AAV capsids may be selected from anAAV which targets CNS, specific cells within the CNS, and/or specificcancer-associated antigens or receptors. Suitable AAV may include, e.g,AAV9 [U.S. Pat. No. 7,906,111; US 2011-0236353-A1], rh10 [WO2003/042397] and/or hu37 [see, e.g., U.S. Pat. No. 7,906,111; US2011-0236353-A1]. However, other AAV, including, e.g., AAV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV7, AAV8 [U.S. Pat. No. 7,790,449; U.S. Pat. No.7,282,199] and others such as, e.g., those described in a word seems tobe missing here may be selected for preparing the AAV vectors describedherein.

Uses and Regimens

Suitably, the composition of the invention are designed so that AAVvectors carry the nucleic acid expression cassettes encoding theimmunoglobulin constructs and regulatory sequences which directexpression of the immunoglobulin thereof in the selected cell. Followingadministration of the vectors into the CNS, the vectors deliver theexpression cassettes to the CNS and express the proteinaceousimmunoglobulin constructs in vivo. The use of compositions describedherein in an anti-neoplastic method are described, as are uses of thesecompositions in anti-neoplastic regimens, which may optionally involvedelivery of one or more other anti-neoplastic or other active agents.

As stated above, a composition may contain a single type of AAV vectoras described herein which contains the expression cassette fordelivering the anti-neoplastic immunoglobulin construct in vivo.Alternatively, a composition may contain two or more different AAVvectors, each of which has packaged therein different expressioncassettes. For example, the two or more different AAV may have differentexpression cassettes which express immunoglobulin polypeptides whichassemble in vivo to form a single functional immunoglobulin construct.In another example, the two or more AAV may have different expressioncassettes which express immunoglobulin polypeptides for differenttargets, e.g., two provide for two functional immunoglobulin constructs(e.g., an anti-Her2 immunoglobulin construct and a secondanti-neoplastic immunoglobulin construct). In still another alternative,the two or more different AAV may express immunoglobulin constructsdirected to the same target, wherein one of the immunoglobulinconstructs has been modified to ablate FcRn binding and a secondimmunoglobulin construct which retains its ability or has enhancedability to bind to FcRn. Such a composition may be useful tosimultaneously provide antibodies with increased retention in the brainarea and antibodies for systemic delivery of the immunoglobulinconstruct.

Optionally, one or both of these immunoglobulin constructs describedherein has enhanced ADCC activity. A regimen as described herein maycomprise, in addition to one or more of the combinations describedherein, further combination with one or more of an anti-neoplasticbiological drug, an anti-neoplastic small molecule drug, achemotherapeutic agent, immune enhancers, radiation, surgery, and thelike. A biological drug as described herein, is based on a peptide,polypeptide, protein, enzyme, nucleic acid molecule, vector (includingviral vectors), or the like.

Suitably, the compositions described herein comprise an anti-neoplasticeffective amount of one or more AAV suspended in a pharmaceuticallysuitable carrier designed for delivery to the subject via injection,osmotic pump, intrathecal catheter, or for delivery by another device orroute. In one example, the composition is formulated for intrathecaldelivery. As used herein, intrathecal delivery encompasses an injectioninto the spinal canal, more specifically into the subarachnoid space.However, other routes of delivery may be selected and thepharmaceutically acceptable carriers for the AAV compositions including,e.g., intracranial, intranasal, intracisternal, intracerebrospinal fluiddelivery, among other suitable direct or systemic routes, i.e. Ommayareservoir.

The compositions can be formulated in dosage units to contain an amountof AAV that is in the range of about 1×10⁹ genome copies (GC) to about5×10¹³ GC (to treat an average subject of 70 kg in body weight). In oneembodiment, a spinal tap is performed in which from about 15 mL (orless) to about 40 mL CSF is removed and in which vector is admixed withthe CSF and/or suspended in a compatible carrier and delivered to thesubject. In one example, the vector concentration is about 3×10¹³ GC,but other amounts such as about 1×10⁹ GC, about 5×10⁹ GC, about 1×10¹⁰GC, about 5×10¹⁰ GC, about 1×10¹¹ GC, about 5×10¹¹ GC, about 1×10¹² GC,about 5×10¹² GC, or about 1.0×10¹³ GC.

The rAAV, preferably suspended in a physiologically compatible carrier,may be administered to a human or non-human mammalian patient. Suitablecarriers may be readily selected by one of skill in the art in view ofthe indication for which the transfer virus is directed. For example,one suitable carrier includes saline, which may be formulated with avariety of buffering solutions (e.g., phosphate buffered saline). Otherexemplary carriers include sterile saline, lactose, sucrose, maltose,and water. The selection of the carrier is not a limitation of thepresent invention. Optionally, the compositions of the invention maycontain, in addition to the rAAV and carrier(s), other conventionalpharmaceutical ingredients, such as preservatives, or chemicalstabilizers.

In one embodiment, the compositions described herein are used in amethod for retarding the growth of a tumor. In still another embodiment,the compositions described herein are useful for decreasing tumor sizein a subject. In a further embodiment, the compositions described hereinare useful in reducing the number of cancer cells in a non-solid tumorcancer. In another embodiment, a composition as provided herein is usedin a method for increasing overall survival and/or progression-freesurvival in a patient. For example, the data in the Examples belowdemonstrates a 33% increase in survival rate in metastatic breast cancerin brain as a solo therapy over the period tested. However, even moremodest increases in survival rate would be desirable. Theanti-neoplastic immunoglobulin constructs are selected with a view tothe neoplasm to be treated. For example, for treatment of a metastaticbreast cancer in the brain, one may engineer an expression cassette foran anti-HER antibody into a recombinant AAV as described herein.Optionally, the AAV compositions as described herein are administered inthe absence of an additional extrinsic pharmacological or chemicalagent, or other physical disruption of the blood brain barrier.

In a combination therapy, the AAV-delivered immunoglobulin constructdescribed herein is administered before, during, or after commencingtherapy with another agent, as well as any combination thereof, i.e.,before and during, before and after, during and after, or before, duringand after commencing the anti-neoplastic therapy. For example, the AAVcan be administered between 1 and 30 days, preferably 3 and 20 days,more preferably between 5 and 12 days before commencing radiationtherapy. In another embodiment of the invention, chemotherapy isadministered concurrently with or, more preferably, subsequent toAAV-mediated immunoglobulin (antibody) therapy. In still otherembodiments, the compositions of the invention may be combined withother biologics, e.g., recombinant monoclonal antibody drugs,antibody-drug conjugates, or the like. Further, combinations ofdifferent AAV-delivered immunoglobulin constructs such as are discussedabove may be used in such regimens.

Any suitable method or route can be used to administer an AAV-containingcomposition as described herein, and optionally, to co-administeranti-neoplastic agents and/or antagonists of other receptors. Theanti-neoplastic agent regimens utilized according to the invention,include any regimen believed to be optimally suitable for the treatmentof the patient's neoplastic condition. Different malignancies canrequire use of specific antitumor antibodies and specificanti-neoplastic agents, which will be determined on a patient to patientbasis. Routes of administration include, for example, systemic, oral,intravenous, intraperitoneal, subcutaneous, or intramuscularadministration. The dose of antagonist administered depends on numerousfactors, including, for example, the type of antagonists, the type andseverity tumor being treated and the route of administration of theantagonists.

The following examples are illustrative only and are not a limitation onthe invention described herein.

EXAMPLES Example 1 CNS Expression of AAV9-Mediated Delivery of GFP

Both GFP and mAbs have been expressed in the CNS of cynomolgus macaquesfollowing intracisternal injection of AAV9 vectors containing a GFPtransgene under either CMV or CB7 promoters at 5×10¹² genome copies(gc)/kg. After 14 days, macaques were necropsided and histology andbiodistribution studies were performed. Post-necropsy histologicalanalysis showed broad CNS expression of GFP in cerebrum, cerebellum,choroid plexus, meninges, and spinal cord ventral horn.

In addition, 3×10¹² gc/kg of AAV9 vector containing the 201 anti-SIVimmunoadhesin (201IA) transgene under the control of the CB7 promoterwas injected intracisternally and CSF was taken at regular intervals tomeasure the concentration of the immunoadesin. The resulting level of201IA expressed in the CSF peaked at ˜600 ng/mL, plateaued at ˜250ng/mL, and remained stable at 198 days post-injection.

A. 2011a Expression Construct

The codon-optimized nucleotide sequence for rhesus macaque anti-SIVmac251 gp120 IgG-201 (Glamann et al. J Virol. 1998; 74(15):7158-7163.doi:10.1128/JVI.74.15.7158-7163.2000.Updated.) immunoadhesin (201IA) wascloned into an AAV expression construct. The construct was flanked byAAV2 inverted terminal repeats and contained a CB7 promoter, a chimericintron, and a rabbit globin polyadenylation sequence(pAAV.CB7.CI.201IA.rBG).

B. I253a Mutation of 201IA to Abrogate FcRn Binding

A nucleotide sequence 768 bp in length complementary to the 201IA genebut containing a mutation corresponding to I253A [SEQ ID NO: 24 providesthe CH2.CH3 fragment with this mutation] or H453A [SEQ ID NO: 23provides the CH2.CH3 fragment with this mutation] of the heavy chainamino acid sequence (Kabat numbering) was obtained from GeneArt (LifeTechnologies). The sequence was flanked by Pst1 and BstZ17I restrictionsites matching those in pAAV.CB7.CI.201IA.rBG. The mutated sequenceswere separately cloned into a pAAV.CB7.CI.201IA.rBG by restrictiondigest using the enzymes indicated (NEB) and ligation (TaKaRa Inc.) asdescribed by the manufacturers. Sanger sequencing (GeneWiz) was used toconfirm complementarity of pAAV.CB7.CI.201IA.rBG [SEQ ID NO: 5 (SEQ IDNO: 6 corresponds to encoded 201IA sequence)],pAAV.CB7.CI.201IA(I253A).rBG [SEQ ID NO: 7 (encoding SEQ ID NO:8)] andpAAV.CB7.CI.201IA(H435A).rBG [SEQ ID NO: 9 (encoding SEQ ID NO:10] oneither side of the desired mutation.

B. IA expression in HEK293 cells and purification by protein A 3×10⁸HEK293 cells (293 cells) were seeded in a 10-stack Cell STACK® (Corning)in DuLbecco's Modified Eagle's Medium (DMEM, Corning CellGro)supplemented with 10% FBS and 1% penicillin/streptomycin (DMEM complete)and incubated at 37° C. 5% CO₂ for 48 hours. 1 mg ofpAAV.CB7.CI.201IA.rBG or pAAV.CB7.CI.201IA(I253A).rBG in TE buffer(Qiagen) was diluted in 42 mL room-temperature antibiotic and serum-freeDMEM. 2 mL PEI-Max 40 KDa, linear (Polysciences) at 1 mg/mL and pH 7.1was diluted separately in 42 mL room-temperature antibiotic andserum-free DMEM. Diluted DNA and diluted PEI were combined and incubatedfor 15 minutes at room temperature. The DNA-PEI mixture was added to 1 Lfinal volume of antibiotic and serum-free DMEM. 293T cells were washedtwice with sterile PBS. The DNA-PEI DMEM mixture was added and incubatedwith the cells for 72 hours at 37° C. 5% CO₂. Supernatant was harvestedand centrifuged for 10 minutes at 3000×g to pellet cellular debris.Supernatant was then concentrated using Centricon® Plus—70 CentrifugalFilter Units (EMD Millipore) according to manufacturer's instructions.201IA or 201IA(I253A) was then purified using a Protein A AntibodyPurification Kit (Sigma) and quantified using a NanoDrop 2000 (ThermoScientific). The purified IAs were then diluted to 1 mg/mL usingglycerol and stored at −20° C.

C. SDS-PAGE/Western Blot Analysis of as

SDS-PAGE using NuPage reagents (Life Technologies) was performedaccording to the manufacturer's instructions. Briefly, 1 μg 201IA,201IA(I253A), or 201IA(H453A) was purified from 293 supernatant or 201IApreviously purified in-house was mixed with NuPage Sample Buffer andNuPage Reducing Agent and heated at 70° C. for 10 minutes. PrecastNuPage 4-12% Bis-Tris 1 mm acrylamide gels were loaded with samples andMagicMark XP Western Protein Standard (LifeTechnologies) and run in 1×NuPage MOPS SDS Running Buffer at 200V for 1 hour. The Trans-Blot®Turbo™ lx transfer system (BioRad) was used to transfer proteins to LFPVDF membranes. Ion reservoir stacks were wetted with 1× Trans-Blot®Turbo™ (TBT) transfer buffer for 2-3 minutes. Pre-cut LF PVDF membraneswere immersed in 100% ethanol until translucent, then transferred to1×TBT buffer for 2-3 minutes. The transfer stack was assembled and runat 1.3 A and 25 V for 7 minutes. The LF PVDF membrane was blockedovernight with gentle shaking in 1×NET buffer+2% gelatin (50 mM Tris HCLpH 7, 125 mM NaCl, 5 mM EDTA pH 8, 0.05% Triton X-100, 2% gelatin indouble distilled H2O). Goat anti-human IgG polyclonal antibodyconjugated to biotin (Abcam) was diluted in 1×NET+2% gelatin andincubated with the membrane at room temperature, washed with 1×NET,incubated with streptavidin-horseradish peroxidase (Abcam) diluted in1×NET+2% gelatin, and washed with 1×NET. The Western blot was detectedusing SuperSignal® West Pico Chemiluminescence Substrate (ThermoScientific) according to the manufacturer's protocol. Images werecaptured using the BioRad ChemiDoc™ MP Image System (Thermo Scientific)with high resolution chemiluminescence automatic settings.

D. 201IA ELISA

All procedures were conducted at room temperature unless indicatedotherwise. Plates were washed with BioTek 405TS microplate washer usingPBS+0.05% Tween-20. mac251 gp120 (Immune Technology Corp.) diluted to 2μg/mL in PBS was incubated overnight on Costar® 96-well EasyWash™ ELISAassay plates (Corning) at 4° C. Plates were then blocked 201IA ELISAblocking buffer (PBS+5% heat-inactivated fetal bovine serum+1 mMEDTA+0.07% Tween-20). Diluted samples were added to plates and diluted2-fold down the plate at least four times. Plates were incubated for 1 hat 37° C. and blocked again in 201IA ELISA blocking buffer. Plates werethen incubated with AffiniPure polyclonal goat anti-human IgG-biotin(Jackson ImmunoResearch Labs) diluted in PBS then withstreptavidin-horseradish peroxidase (Abcam) diluted in PBS.3,3′,5,5′-tetramethylbenzidine (TMB) substrate was used to develop theplates. After stopping the colorimetric reaction with H₂ SO₄, plateswere read using a SpectraMax M3 (Molecular Devices) plate reader at 450nm.

Equivalent performance of 201IA or 201IA(I253A), and performance of201(H453) purified from 293 cells as described in these examples and a201IA standard protein produced in-house was determined by 201IA ELISA.Each IA was diluted to 50 ng/mL and assayed as described above. The201IA used as a standard was produced as follows. RAG KO mice wereinjected with an AAV8.TBG.201IA vector at 3×10¹¹ GC/per mouseintravenously, and orbital bleeds were collected on a weekly basis for 8weeks, mice terminated by cardiac bleed. There are generally 5 mice pergroup. All collected serum is pooled together and loaded on the proteinA affinity column from SIGMA as described above. Generally, purified201IA is diluted to 1 mg/ml and glycerol added so that final glycerol isabout 20% for better storage.

E. AAV9 Vector Production

pAAV.CB7.CI.201IA.rBG and pAAV.CB7.CI.201IA(I253A).rBG were packaged inan AAV9 capsid by triple transfection of 293 cells and purified aspreviously described in M. Lock et al, Hum Gene Ther. 2010 October;21(1); 1259-1271, published online 2010 Sep. 24.

F. Expression of 201IA and 201IA(I253A) in Brain and Serum of Mice

All animals were maintained according to NIH and USDA guidelines for thecare and use of animals in research. 6-8 week-old female Rag1−/−(Jackson Labs #002216), FcRn−/− Rag1−/− (Jackson Labs #017700), or humanFcRn transgenic mice (mFcRn−/− hFcRn+/+, Jackson Labs #016919) on aC57BL/6 background were obtained and kept at the University ofPennsylvania.

For vector administration, AAV9.CB7.CI.201IA.rBG orAAV9.CB7.CI.201IA(I253A).rBG was diluted in sterile PBS. For intravenous(IV) administration, vector was diluted to 1×10¹⁰ or 1×10¹¹ genomecopies (GC) per 100 μL. For intracerebroventricular (ICV)administration, vector was diluted to 1×10¹⁰ GC or 1×10¹¹ GC per 10 μL.IV injection was performed by tail-vein injection, and ICV injection wasperformed free-hand as described previously (Glascock et al. J Vis Exp.2011 Oct. 3; (56)) after isofluorane induction of anesthesia. Blood wastaken at days 3, 7, 14, 21, 28, 42, 56, and a final time point of day 60for 201IA(I253A) or day 76 for 201IA by retro-orbital bleed into ZGel™microtube serum separators (Sarstedt). Blood was incubated at roomtemperature for 20 minutes then centrifuged for 5 minutes at 5000×g.Serum was kept at −80° C. and used in the 201IA ELISA described above inPart D to measure serum 201IA concentration.

At necropsy, mice were deeply anesthetized with 100 mg/kg ketamine and10 mg/kg xylazine in sterile PBS to a spinal plane of anesthesia. Thethoracic cavity was exposed. A 20 gauge Angiocath™ Autoguard™ IVcatheter (Becton Dickenson) was inserted into the left ventricle of theheart, and the right atrium was nicked with scissors. 50 mL PBS withHeparin (10 U/mL, Sigma) was administered into the left ventricle slowlythrough the IV catheter using a 30 mL hand-held syringe. Fluid exitingthe right atrium was clear at the end of the perfusion procedure. Brain,liver, and spleen were removed and frozen immediately on dry ice. Braintissue extract was prepared by quartering frozen mouse brains (˜100 mgbrain per quarter) and immersing them in 1 mL tissue lysis buffer (25 mMTris-HCl, 5 mM EDTA, 1% Triton™-X, 150 mM NaCl, pH 7.6). Samples werehomogenized with stainless-steal beads using a TissueLyzer™ (Qiagen) at30 Hz for 2 minutes, frozen overnight at −80° C., thawed in aroom-temperature water bath, and centrifuged at 10K×g for 10 minutes at4° C. Supernatants from each of the four sections of an individual mousebrain were combined. After gentle vortexing, the brain extracts werealiquoted and frozen at −80° C. until use. Diluted brain extracts wereused in the 201 IA ELISA described above to determine 201IA expressionin brain.

Serum expression of each the I253A and the H435A 201IA mutants wassignificantly lower than the wild-type 201IA (standard) after both iv oricy administration at both tested doses (1×10¹⁰ GC/mouse or 1×10¹¹GC/mouse). The brain extracts tested after ICV administration (1×10¹¹GC/mouse) showed expression of the I253A mutant in the brain at levelsexceeding those of the wild-type (standard). Expression of H435A (1×10¹¹GC/mouse) was observed.

G. Expression of 201IA and 201IA(I253A) in CSF and Serum of CynomolgusMacaques

Four 3-4 year-old female cynomolgus macaques weighing between 3-5 kgwere housed in stainless steel caging on a 12-hour light/dark cycle atthe University of Pennsylvania according to according to NIH and USDAguidelines for the care and use of animals in research. Animals wereacclimatized 7 weeks prior to initiation of studies. Monkeys were givenPrimate Diet 5049 (PMI Feeds Inc.). Water was given ad lib from anautomatic watering system.

On the day of vector administration, animals were anesthetized usingketamine (10-15 mg/kg) and dexmedetomidine (0.05-0.10 mg/kg) givenintramuscularly (IM). Animals were weighed, and vital signs wererecorded. The hair over the back of the head and cervical spine wasshaved. The skin was sterilely prepped with betadine. The neck wasflexed so that the chin was almost touching the chest (care was takennot to occlude the animal's airway). The occipital protuberance at theback of the skull and the wings of the atlas (C1) were palpated and thespinal needle or regular needle (20-24 gauge) was inserted midwaybetween them. If bone was encountered, the needle was redirectedanteriorly or posteriorly. Once in the subarachnoid space, CSF wascollected via gravitational flow into a syringe or other sterilecontainer as it welled up into the hub of the needle (up to 2 mL).Suction was not applied to the needle. Up to 2 mL of vector solution wasinjected using a syringe pump at 0.5 mL/minute or manually at a slowsteady pace. The needle was removed and direct pressure applied to thepuncture site. Two macaques received AAV9.CB7.CI.201IA.rBG.N401 and tworeceived AAV9.CB7.CI.201IA(I253A).rBG. The dose was 1.00×10¹² VG perkilogram of body weight.

At least once every two weeks, animals were monitored for vital signs,clinical pathology, and immunology. Blood and lumbar CSF were collectedat day 8 and 15 after the procedure, then monthly. Serum and CSF wasstored at approximately −65 to −80° C. 201IA expression was evaluated by201IA ELISA as indicated above. Changes in the blood chemistries andblood profiles of the animals were monitored by the contract facilityAntech Diagnostics, Inc.

Monkeys will be euthanized at the end of their study period. The animalsare first sedated with ketamine (10-15 mg/kg) and dexmedetomidine(0.05-0.10 mg/kg) IM. They are euthanized using sodium pentobarbital (80mg/kg IV). Death is confirmed by absence of heartbeat and respiration.The animal may also be exsanguinated to assure death. Collected tissueswill be placed in 10% neutral buffered formalin for histopathology. Forgenome copy analysis, tissue samples will be immediately frozen on dryice and maintained at <−60° C. Samples will be directly frozen in OCTembedding medium for cryosectioning. Slides will be prepared by Cellularand Morphology Core of the Gene Therapy Program of the University ofPennsylvania. Other appropriate stains may be employed at the discretionof the study pathologist.

Example 2 Production of AAV9 Expressing Trastuzumab

A well-published murine xenograft model of breast cancer brainmetastasis is used to determine if trastuzumab expressed in the CNSprolongs survival or alleviates tumor burden [Martinez-Aranda A, et al,Development of a Preclinical Therapeutic Model of Human Brain Metastasiswith Chemoradiotherapy. Int J Mol Sci. 2013; 14:8306-8327]. HER2positive human BT474 ductal carcinoma cells are transfected withluciferase and injected stereotactically into the brain parenchyma ofnude mice. Tumor size will be monitored by luminescent intensity. Whenthe tumors grow to 10 mm², the mice will be injected intraventricularlywith varying concentrations of AAV9 vector carrying a trastuzumabtransgene.

The transgene is created by cloning the codon-optimized nucleic acidsequences now provided in SEQ ID NO: 1, which encode the publishedsequences of the light and heavy variable chains of trastuzumab, into anIgG expression cassette. The constant region amino acid sequencesdescribed in WO 2015/012924, which is incorporated by reference herein,can be used. See, e.g., Carter P, et at, Humanization of ananti-p185HER2 antibody for human cancer therapy. Proc Natl Acad Sci USA.1992 May 15; 89(10):4285-9 describing humanization of the murine mAbprecursor of trastuzumab. These amino acid sequences exactly match thoseof the clinical product sequenced by mass spectrometry in 2013 [GahoualR, et al, Rapid and multi-level characterization of trastuzumab usingsheathless capillary electrophoresis-tandem mass spectrometry. MAbs.2013 Apr. 5; 5(3). [Epub ahead of print]. After injection of the vector,tumor size and mouse survival will be monitored for 30 days. At necropsypathological examination of the tumor will be conducted and level oftrastuzumab expression by ELISA of brain extracts determined. The vectorand method described herein should provide prolonged survival,progression-free survival, and/or regression and/or stabilization oftumor burden.

A. Trastuzumab Expression Construct

Sequences matching the WHO published nucleotide sequences of the heavyand light chains of trastuzumab were obtained from GeneArt (LifeTechnologies). The light chain sequence was flanked by EcoRV and BsiW1restriction sites, and the heavy chain sequence was flanked by Xba1 andSal1 restriction sites [the nucleic acid sequence is provided in SEQ IDNO: 11, which encodes trastuzumab heavy chain variable (SEQ ID NO: 12),heavy chain constant (SEQ ID NO: 13), light chain variable (SEQ ID NO:14), kappa chain (SEQ ID NO: 15), and Amp-R (SEQ ID NO: 16)] providesthe sequences of the plasmids containing the trastuzumab heavy and lightchains.

The heavy and light chain sequences were cloned into an AAV expressionconstruct using restriction digest (NEB) and ligation (TaKaRa Inc.)using known cloning techniques. The heavy and light chain sequences wereseparated from each other by an F2A self-cleaving peptide. The constructwas flanked by AAV2 inverted terminal repeats and contained a CMVimmediate early promoter, a chimeric intron, and a SV40 polyadenylationsignal, termed pAAV.CMV.CI.trastuzumab.SV40 [SEQ ID NO: 17, encodingtrastuzumab heavy chain variable, heavy chain constant, light chainvariable, kappa chain (SEQ ID NO: 18-21, respectively)].

B. Trastuzumab Expression in HEK293 Cells and Purification by Protein A

3×10⁸ HEK293 cells [obtained from the ATCC] were seeded in a 10-stackCellSTACK® in DMEM complete at 37° C. 5% CO₂ for 48 hours. 1 mg ofpAAV.CMV.CI.trastuzumab.SV40 (described in Part A) was diluted in 42 mLroom-temperature antibiotic and serum-free DMEM. 2 mL PEI-Max 40 KDa,linear (Polysciences) at 1 mg/mL and pH 7.1 was diluted separately in 42mL room-temperature antibiotic and serum-free DMEM, Diluted DNA anddiluted PEI were combined and incubated for 15 minutes at roomtemperature. The DNA-PEI mixture was added to a 1 L final volume ofantibiotic and serum-free DMEM. 293 cells were washed twice sterile PBS,and the cells were then incubated with the DNA-PEI DMEM mixture for 72hours. Supernatant was harvested, centrifuged for 10 minutes at 3000×gto pellet cellular debris, and concentrated using Centricon® Plus—70Centrifugal Filter Units (EMD Millipore) according to manufacturer'sinstructions. Trastuzumab was then purified using a Protein A AntibodyPurification Kit (Sigma) and quantified using a NanoDrop 2000 (ThermoScientific). The purified trastuzumab was diluted to 1 mg/mL usingglycerol and stored at −20° C.

C. SDS-PAGE/Western Blot Analysis of IAs

SDS-PAGE using NuPage reagents (Life Technologies) was performedaccording to the manufacturer's instructions. Briefly, 1 μg trastuzumabpurified from 293 supernatant as described in Part B of this Example ortrastuzumab clinical product resuspended in PBS (Hoffmann-La Roche, HUPPharmacy), was mixed with NuPage Sample Buffer and NuPage Reducing Agentand heated at 70° C. for 10 minutes. Precast NuPAGE® 4-12% gradientBis-Tris (neutral pH) 1 mm acrylamide gels were loaded with samples andMagicMark™ XP Western Protein Standard (LifeTechnologies) and run in 1×NuPage® 3-morpholinopropane-1-sulfonic acid (MOPS) SDS Running Buffer at200V for 1 hour. The Trans-Blot® Turbo™ 1× transfer system (BioRad) wasused to transfer proteins to low fluorescence (LF) polyvinylidenefluoride (PVDF) membranes. Ion reservoir stacks were wetted with 1×Trans-Blot® Turbo™ (TBT) transfer buffer for 2-3 minutes. Pre-cut LFPVDF membranes were immersed in 100% ethanol until translucent, thentransferred to 1×TBT buffer for 2-3 minutes. The transfer stack wasassembled and run at 1.3 A and 25 V for 7 minutes. The LF PVDF membranewas blocked overnight with gentle shaking in 1×NET buffer+2% gelatin (50mM Tris HCl pH 7, 125 mM NaCl, 5 mM ethylenediaminetetraacetic acid(EDTA) pH 8, 0.05% Triton™ X-100 (Triton™ X-100 is a nonionic surfactantthat has a hydrophilic polyethylene oxide chain and an aromatichydrocarbon lipophilic or hydrophobic group), 2% gelatin in doubledistilled H₂O). Goat anti-human IgG polyclonal antibody conjugated tobiotin (Abcam) was diluted in 1×NET+2% gelatin and incubated with themembrane at room temperature, washed with 1×NET, incubated withstreptavidin-horseradish peroxidase (Abcam) diluted in 1×NET+2% gelatin,and washed with 1×NET. The Western blot was detected using SuperSignal®West Pico Chemiluminescence Substrate (Thermo Scientific) according tothe manufacturer's protocol. Images were captured using the BioRadChemiDoc™ MP Image System (Thermo Scientific) with high resolutionchemiluminescence automatic settings.

D. Vector Production

pAAV.CMV.CI.trastuzumab.SV40 was packaged in an AAV9 capsid by tripletransfection of 293 cells and purified as previously described (Lock etal, 2010, cited above).

E. Trastuzumab ELISA

A trastuzumab mimotope ELISA was developed as described previously(Jiang et al. J Biol Chem. 2005 Feb. 11; 280(6):4656-62. Epub 2004 Nov.9). All steps were performed at room temperature unless otherwisestated. Plates were washed with a BioTek 405TS microplate washer. Apeptide mimotope of the epitope of HER2 to which trastuzumab binds,LLGPYEL WELSH [SEQ ID NO: 22], was obtained from the mimotopes,resuspended in DMSO, and stored at −80° C. Costar® 96-well EasyWash™ELISA assay plates (Corning) were coated at 1 μg/mL LLGPYELWELSH [SEQ IDNO: 22] in 100 mM bicarbonate solution (pH 9.6), incubated overnight at4° C., and blocked with trastuzumab ELISA blocking buffer (TEB, PBS+5%bovine serum albumin+1 mM EDTA+0.07% Tween-20). Samples were diluted inTEB and plated, diluted 2-fold down the ELISA plate in TEB, andincubated. Plates were then incubated with AffiniPure polyclonal goatanti-human IgG-biotin (Jackson ImmunoResearch Labs) diluted in TEBfollowed by streptavidin-horseradish peroxidase (Abcam) diluted in TEB.Plates were developed with TMB substrate, stopped with 2N H₂SO₄, thenread using a SpectraMax M3 (Molecular Devices) plate reader at 450 nm.

F. Expression of 201IA and 201IA(I253A) in Brain and Serum of Mice

6-8 week-old female Rag1−/− mice (Jackson Labs #002216) on a C57BL/6background were obtained and kept at the University of Pennsylvania andmaintained according to NIH and USDA guidelines for the care and use ofanimals in research.

For vector administration, pAAV.CMV.CI.trastuzumab.SV40 was diluted insterile PBS. For intravenous (IV) administration, vector was diluted to1×10¹⁰ or 1×10¹¹ GG per 100 μL. For intracerebroventricular (ICV)administration, vector was diluted to 1×10¹⁰ or 1×10¹¹ GC per 10 μL. IVinjection was performed by tail-vein injection, and ICV injection wasperformed free-hand after isofluorane induction of anesthesia asdescribed previously (Glascock et al.). Blood was taken at days 3, 7,14, 21, 28, 42, 56, and 60 post-vector administration by retro-orbitalbleed into Z-Gel microtube serum separators (Sarstedt). Blood wasincubated at room temperature for 20 minutes then centrifuged for 5minutes at 5000×g. Serum was kept at −80° C. and used in the trastuzumabELISA described above to measure serum trastuzumab concentration.

At necropsy, mice were deeply anesthetized with 100 mg/kg ketamine and10 mg/kg xylazine in sterile PBS to a spinal plane of anesthesia. Thethoracic cavity was exposed. A 20 gauge Angiocath™ Autoguard™ IVcatheter (Becton Dickenson) was inserted into the left ventricle of theheart, and the right atrium was nicked with scissors. 50 mL PBS withHeparin (10 U/mL, Sigma) was administered into the left ventricle slowlythrough the IV catheter using a 30 mL hand-held syringe. Fluid exitingthe right atrium was clear at the end of the perfusion procedure. Brain,liver, and spleen were removed and frozen immediately on dry ice.

Brain tissue extract was prepared by quartering frozen mouse brains(˜100 mg brain per quarter) and immersing them in 1 mL tissue lysisbuffer (25 mM Tris-HCl, 5 mM EDTA, 1% Triton-X, 150 mM NaCl, pH 7.6).Samples were homogenized with stainless-steal beads on a TissueLyzer(Qiagen) at 30 Hz for 2 minutes, frozen overnight at −80° C., thawed ina room-temperature water bath, and centrifuged at 10K×g for 10 minutesat 4 C. Supernatants from each of the four sections of a single mousebrain were combined. After gentle vortexing, the brain extract wasaliquotted and frozen at −80° C. until use. Diluted brain extracts wereused in the trastuzumab ELISA above to measure brain trastuzumabconcentration.

These data show the steady state expression levels of >1000 μg/mL in theserum of Rag1−/− mice following intravenous vector delivery for theduration of the experiment (60 days). For the mice receiving ICV vectoradministration, the steady state expression level of >750 μg/mL isobserved for the duration of the experiment (60 days). These amounts arebelieved to demonstrate expression of levels which will provide atherapeutic effect.

The brain studies revealed concentrations of between about 1200 to about1800 μg trastuzumab in the test mice for those receiving 1×10¹⁰ and1×10¹¹ intravenous vector. There did not appear to be any significantdifference between these dosage levels for iv delivery. At the samedoses, greater variation was observed for the vectors delivered at theseconcentrations via ICV. The concentrations varied from about 1000 μg toabout 2500 μg.

G. Generation of HER2+BT474-M1 Breast Cancer Cell Line ExpressingFirefly Luciferase

HER2+BT474.M1 human ductal carcinoma cells at passage 27 were a generousgift from Louis Chodosh and Jason Ruth. [BT474.MI cells is a subclone ofBT474 that can be obtained from California Pacific Medical Center; SiTuen Lee-Hoeflich, et al., Cancer Res Jul. 15, 2008 68; 5878.] Cellswere grown in a T75 tissue culture flask (Corning) in DMEM/F12 media(Corning Cellgro) supplemented with 10% FBS and 1%penicillin/streptomycin (DMEM/F12 complete).VSVG.HIV.SIN.cPPT.CMV.ff-luciferase.WPRE lentiviral vector was obtainedfrom the Penn Vector Core [E. Coprini et al, Viruses, August 2010, 2(8):1577-1588.] When BT474-M1 cells were 60-70% confluent, media wasaspirated, cells were washed with sterile PBS, trypsinized, and counted.2.5×10⁵ cells in 2 mL DMEM/F12 complete were added to the wells of asix-well tissue culture treated plate (Falcon) and incubated overnightat 37° C. 5% CO₂. Vector was diluted in antibiotic and serum freeDMEM/F12 to 3.5×10⁸ VG/mL, and five 2-fold serial dilutions wereprepared. 1 mL of the six vector dilutions were added to correspondingwells of the 6-well plate containing PBS-washed BT474-M1 cells and 1 mLantibiotic and serum-free DMEM/F12. The plate was incubated for 48 hoursat 37° C. 5% CO₂. Media was aspirated and replaced with DMEM/F12complete. No cytopathology was noted by microscopy. After another 72hours, the cells in the 3 wells that received the highest concentrationof vector were washed with sterile PBS, trypsinized, mixed, and culturedin a T75 flask in DMEM/F12 complete. After 72 hours at 37° C. 5% CO₂,cells were trypsinized and diluted to a concentration of 1 cell per 200μL DMEM/F12 complete. 200 μL cell suspension per well was plated in a96-well tissue culture-treated plate (Falcon) and incubated at 37° C. 5%CO₂ for 6 weeks. Media was changed every 2 weeks. Two weeks afterplating, wells with single colonies of clonal cells were noted bymicroscopy. When wells were 70% confluent with a single cluster ofclonal cells, fifteen clones were selected for further expansion to70-80% confluency in 24-well plates, then in 6-well plates, then T25tissue culture flasks (Corning). Morphology of the cells was comparedwith the parental BT474.M1 cell line and noted to be equivalent.

Luciferase activity of the cells was measured using the Dual Luciferase®Reporter Assay System (Promega) to the manufacturer's instructions. DNAwas isolated from cells using the DNeasy Kit (Qiagen). Copy number ofluciferase per cell in the five clones with the highest luminescence wasdetermined by TaqMan Real Time PCR (Life Technologies) using thelentiviral packaging signal as a probe [A Hachiya et al, Gene Ther,April 2007; 14(8): 648-656, Epub 2007 Feb. 1]. The clone chosen forxenograft experiments was expanded to passage number 52 andcryopreserved in liquid nitrogen in DMEM/F12 with 5% DMSO and 20% FBS.

H. Xenograft Model of HER2+ Breast Cancer Brain Metastases in Rag1−/−Mice

BT474-M1.ffluc cells prepared as described in Part G of this examplewere thawed, washed in DMEM/F12 complete, expanded in T175 flasks(Corning) at 37° C. 5% CO₂ in DMEM/F12 complete, and passaged once atleast 1 week before tumor cell implantation. On the day of injection,cells at passage 53 were trypsinized at 70%-80% confluency and countedusing a hemacytometer. Cells were centrifuged for 3 minutes at 1000×gand washed with sterile PBS. After centrifuging again, cells wereresuspended at 1×10⁵ cells/1 μL in sterile PBS and kept on ice untilinjection. For the tumor cell injection procedure, mice wereanesthetized by intraperitoneal (IP) injection of 100 mg/kg ketamine and10 mg/kg xylazine in sterile PBS to induce a spinal plane of anesthesia.Opthalmic ointment was applied to the eyes of the mice ad lib. Hair wassheared from the top of the mouse's head using electric clippers.Estrogen pellets (1.6 mg, 60-day release) were implanted subcutaneouslyby cleansing the exposed skin first with povidone iodine then 70%ethanol. A small incision in the skin overlaying the thoracic spine wasmade, and the skin and underlying fascia were bluntly dissected. Theestrogen pellet was implanted subcutaneously, and the incision wassutured with 4.0 vicryl. Next, the mice were then fixed in astereotactic apparatus. The exposed skin over the skull was cleaned withpovidone iodine followed by 70% ethanol. An anterior-posterior incisionapproximately 1 cm long was made over the top of the skull with a 22scalpel blade. Bregma was identified. A pneumatic drill was positionedat bregma and coordinates were noted. The drill point was moved −0.8 mmanterior-posterior, +2.2 mm mediolateral of bregma, and a burr hole wasdrilled until brain parenchyma was reached. The drill was removed fromthe stereotactic apparatus, and a 10 μL Hamilton syringe was loaded with1 μL of cell suspension. The needle was positioned on the apparatus,brought to bregma, and moved to the coordinates indicated above. Theneedle was checked for exact positioning over the burr hole, andcoordinates were adjusted accordingly before penetrating −4.0 mm DV ofbregma, then +1.0 mm. 1 μL of cell suspension was injected over 5minutes. The needle was left in place for 5 minutes after injection,then removed slowly. The mouse was removed from the stereotacticapparatus, and 4.0 vicryl was used to suture the incision over theskull. Mice were placed in a clean cage on top of a heating pad set to37° C. After recovering from anesthesia, the mice were given 100 μL of15 mg/kg enrofloxacin (Bayer) in sterile PBS along with 0.3 mg/kgbuprenorphine in sterile PBS subcutaneously. Mice received enrofloxacinsubcutaneously for two days after the procedure.

Growth of tumor was monitored every 3-4 days using bioluminescentimaging (BLI). Mice were injected IP first with 150 mg/kg luciferin insterile PBS then with 100 mg/kg ketamine and 10 mg/kg xylazine in PBSfive minutes later. 5-10 minutes after anesthesia was administered, micewere imaged using an IVIS Xenogen imaging system. Bioluminescence wasmeasured for at least 5 seconds. Regions of interest (ROI) correspondingto luminescent tumors were measured by drawing a gate around the ROI.Luminescence was reported in photons/second. At necropsy, mice wereeuthanized by overexposure to CO2 followed by cervical dislocation.Brains were removed and preserved in formalin followed by 70% ethanoland embedded in paraffin for sectioning. Hematoxylin and eosin as wellas luciferin immunostaining was performed. Liver and spleen were alsotaken at necropsy for biodistribution analysis of vector genomes.

I. Prophylactic Treatment of Xenograft Model of Breast Cancer BrainMetastases

6-8 week-old female Rag1−/− mice (Jackson Labs #002216) were treated 21days prior to tumor implantation with an ICV injection of 1×10^(1I) VGof AAV9.CMV.CI.trastuzumab.SV40 (n=9), AAV9.CB7.CI.201IA.rBG (n=10), orno treatment (n=5). Tumors were implanted and bioluminescence wasmeasured as indicated above. Blood was taken retro-orbitally from miceon D20, 36, 62, and 72 post vector injection to measure serumtrastuzumab as a surrogate for CNS trastuzumab expression. Afterreaching a tumor BLI of 1×10⁸ photons/second, mice are monitored dailyand sacrificed at a clinical endpoint defined as neurological impairmentor significant morbidity including lethargy, hunching, paralysis,neurological deficits, or seizures.

FIG. 2 provides a survival curve of mice given 1×10¹¹ GC ICV ofAAV9.trastuzumab or AAV9.201IA prophylactically, then implanted withBT474-M1.ffluc tumor cells in the brain 21 days after vectoradministration. This curve reflects results 99 days post-tumorimplantation. The median survival of 201IA group (sham treatment) isshown to be 66 days, whereas the median survival of the group treatedwith AAV9.trastuzumab is 99 days, a 33% increase in survival rate.

The biodistribution of the AAV9.trastuzumab as delivered iv and icy inRag1−/− mice was assessed. At a dose of 1×10¹⁰ GC for both iv andicy-delivered vectors, relatively low levels of vector genomes areobserved in either liver or brain. At the higher dose (1×10¹¹ GC),significantly higher levels of vectors are found in liver for bothdelivery methods, whereas significantly higher levels in brain are foundonly in the animals receiving icy administration. It is notable thatthis vector contained a non-tissue specific promoter. Safety concernsmay be reduced via use of a tissue specific promoter which specificallytargets cells of the brain and optionally other neural cells or cells inthe central nervous system, in order to minimize expression in liver.Alternatively, expression in liver may be beneficial for the systemicdelivery of trastuzumab in order to prevent or control breast cancermetastasis into other organs.

J. Alternative Mouse Model Suitable for Studies of BT474-M1 BreastCancer Brain Metastases Using NSG Mice

6-8 week-old female NSG mice (Jackson Labs #005557) were kept at theUniversity of Pennsylvania. Tumors were implanted as indicated abovewith the following changes to the preparation of tumor cells andinjection technique. Tumor cells were resuspended in 50% MatriGel®(Corning)/50% sterile PBS at 1×10⁵ cells per 5 μL. Injection volume wasincreased from 1 μL to 5 μL. After positioning the needle in the brainparenchyma, 5 minutes elapsed before injection began. The injection wasperformed slowly over 10 minutes, and the syringe was left in place 5minutes before removal. Mice were monitored and bioluminescence wasmeasured as indicated elsewhere in this document. The data showedsuccessful engraftment of the tumor cells into brain. The rates of thetumor growth will be evaluated to determine if this model is desirablefor study of breast cancer metastasis to brain.

SEQUENCE LISTING FREE TEXT

The following information is provided for sequences containing free textunder numeric identifier <223>.

SEQ ID NO: (containing free text) Free text under <223> 1 <223>engineered anti-HER antibody <220> <221> misc <222> (1) . . . (60) <223>IL2 signal/leader peptide <220> <221> CDS <222> (61) . . . (423) <223>heavy variable <220> <221> Misc <222> (439) . . . (714) <223> CH1 <220><221> Misc <222> (715) . . . (1410) <223> CH1 <220> <221> Misc <222>(2010) . . . (2069) <223> IL2 signal/leader peptide <220> <221> Misc<222> (2070) . . . (2391) <223> variable light <220> <221> Misc <222>(2407) . . . (2711) <223> light chain constant region 2 <223> SyntheticConstruct 3 <223> engineered anti-HER heavy chain <220> <221>MISC_FEATURE <222> (1) . . . (20) <223> heavy chain signal peptide <220><221> MISC_FEATURE <222> (21) . . . (140) <223> heavy chain variableregion <220> <221> MISC_FEATURE <222> (141) . . . (259) <223> heavychain constant region 1 <220> <221> MISC_FEATURE <222> (260) . . . (470)<223> heavy chain FC 4 <223> Engineered anti-Her2 light chain <220><221> MISC_FEATURE <222> (1) . . . (20) <223> signal sequence <220><221> MISC_FEATURE <222> (21) . . . (130) <223> light chain variableregion <220> <221> MISC_FEATURE <222> (131) . . . (214) <223> lightchain constant region 5 <223> pAAV.CB7.CI.201IA.rBG <220> <221>misc_feature <222> (275) . . . (404) <223> 3′ ITR (complement) <220><221> misc_feature <222> (3226) . . . (3355) <223> 5′ ITR <220> <221>misc <222> (3423) . . . (3804) <223> CMV IE promoter <220> <221> CDS<222> (5161) . . . (6690) <223> 201IA 6 <223> Synthetic Construct 7<223> Plasmid encoding 201IA(I253A)mutant <220> <221> misc <222> (1) . .. (130) <223> 5′ ITR <220> <221> misc_feature <222> (198) . . . (579)<223> CMV IE promoter <220> <221> promoter <222> (582) . . . (863) <223>CB promoter <220> <221> Intron <222> (958) . . . (1930) <223> chickenbeta-actin intron <220> <221> CDS <222> (1936) . . . (3465) <223> CMV IEpromoter <220> <221> polyA_signal <222> (3529) . . . (3655) <223> rabbitglobin polyA <220> <221> misc_feature <222> (3744) . . . (3873) <223> 3′ITR (complement) <220> <221> misc_feature <222> (4636) . . . (5493)<223> AP(R) marker 8 <223> Synthetic Construct 9 <223> engineeredplasmid containing 201IA(H435) mutant <220> <221> misc_feature <222>(275) . . . (404) <223> 3′ ITR (complement) <220> <221> misc_feature<222> (3423) . . . (3804) <223> CMV IE promoter <220> <221> promoter<222> (3807) . . . (4088) <223> CB promoter <220> <221> TATA_signal<222> (4061) . . . (4064) <223> rabbit globin polyA <220> <221> Intron<222> (4183) . . . (5155) <223> chicken beta-actin intron <220> <221>CDS <222> (5161) . . . (6690) <223> CMV IE promoter 10 <223> SyntheticConstruct 11 <223> Plasmid containing the heavy and light chains oftrastuzumab <220> <221> misc_feature <222> (1) . . . (130) <223> 5′ ITR<220> <221> promoter <222> (191) . . . (932) <223> human CMV IE enhancerand promoter <220> <221> sig_peptide <222> (1305) . . . (1364) <223>IL-2 signal peptide <220> <221> CDS <222> (1365) . . . (1724) <223>Trastuzumab heavy variable <220> <221> CDS <222> (1725) . . . (2720)<223> trastuzumab heavy constant <220> <221> enhancer <222> (2726) . . .(3313) <223> IRES <220> <221> sig_peptide <222> (3314) . . . (3373)<223> IL2 signal peptide <220> <221> CDS <222> (3374) . . . (3694) <223>trastuzumab light variable <223> trastuzumab constant light (kappa)<220> <221> polyA_signal <222> (4038) . . . (4269) <223> SV40 late polyAsignal <220> <221> polyA_signal <222> (4083) . . . (4269) <223> SV40late polyA signal <220> <221> misc_feature <222> (4334) . . . (4463)<223> 3′ ITR (complement) <220> <221> rep_origin <222> (4640) . . .(5095)) <220> <221> CDS <222> (5226) . . . (6083) <223> amp-R <220><221> misc_feature <222> (6257) . . . (6845) <223> COL/E1/origin 12<223> Synthetic Construct 13 <223> Synthetic Construct 14 <223>Synthetic Construct 15 <223> Synthetic Construct 16 <223> SyntheticConstruct 17 223> Engineered plasmid containing trastuzumab MAb <220><221> sig_peptide <222> (1254) . . . (1313) <223> IL2 signal sequence<220> <221> CDS <222> (1314) . . . (1673) <223> Trastuzamab heavyvariable <220> <221> CDS <222> (1674) . . . (2660) <223> Trastuzamabheavy constant <220> <221> sig_peptide <222> (2745) . . . (2804) <223>IL2 signal sequence <220> <221> CDS <222> (2805) . . . (3110) <223>Trastuzamab light variable <220> <221> CDS <222> (3111) . . . (3452)<223> Trastuzamab light constant (kappa) 18 <223> Synthetic Construct 19<223> Synthetic Construct 20 <223> Synthetic Construct 21 <223>Synthetic Construct 22 <223> Peptide mimotope of HER2 epitope 23 <223>trastuzumab immunoglobulin H435A mutant 24 <223> immunoglobulin fragmentwith I253A mutant 25 <223> Trastruzumab heavy chain

This application contains sequences and a sequence listing, which ishereby incorporated by reference. U.S. Provisional Patent ApplicationNo. 61/984,646, filed Apr. 25, 20145, and all publications, patents, andpatent applications cited in this application are hereby incorporated byreference in their entireties as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications can be made thereto withoutdeparting from the spirit or scope of the appended claims.

1. A composition comprising at least one AAV vector formulated forcentral nervous system delivery, wherein said composition comprises atleast one expression cassette which contains sequences encoding ananti-neoplastic immunoglobulin construct for delivery to the brainoperably linked to expression control sequences therefor and apharmaceutically acceptable carrier.
 2. The composition according toclaim 1, wherein the anti-neoplastic immunoglobulin construct comprisesan immunoglobulin modified to have decreased or no measurable affinityfor neonatal Fc receptor (FcRn).
 3. The composition according to claim1, wherein the anti-neoplastic immunoglobulin construct comprises animmunoglobulin modified to have increased ADCC activity.
 4. Thecomposition according to claim 2, wherein the immunoglobulin constructhas been modified in one or more of positions Y436 (aa459 of SEQ ID NO:25), S254 (aa277 of SEQ ID NO:25), I253 (aa276 of SEQ ID NO: 25), and/orH435 (aa458 of SEQ ID NO: 25).
 5. The composition according to claim 1,wherein the anti-neoplastic immunoglobulin construct is selected from amonoclonal antibody, an Fv, Fab, F(ab)2, F(ab)3, Fab′, Fab′-SH, F(ab′)2,an immunoadhesin, or a single chain variable fragment antibody.
 6. Thecomposition according to claim 1, wherein the immunoglobulin constructis directed to a primary or metastatic CNS cancer.
 7. The compositionaccording to claim 6, wherein the immunoglobulin is an anti-Her2antibody selected from the group consisting of trastuzumab,212-Pb-TCMC-trastuzumab, or pertuzumab.
 8. The composition according toclaim 7, wherein the sequences encoding the heavy chain of trastuzumabhave the nucleic acid sequence of nt 61-1410 SEQ ID NO: 1, or a sequenceat least 90% identical thereto.
 9. The composition according to claim 7,wherein the sequences encoding the light chain of trastuzumab have thenucleic acid sequence shown in nt 2070-2711 of SEQ ID NO: 1, or asequence at least 90% identical thereto.
 10. The composition accordingto claim 1, wherein the at least one AAV vector has an AAV9, AAV rh10 orAAV hu37 capsid.
 11. The composition according to claim 1, wherein thecomposition comprises AAV comprising at least two different antibodyexpression cassettes.
 12. The composition according to claim 1, whereinthe composition comprises a single antibody expression cassette.
 13. Acomposition comprising an AAV viral vector having an AAV9 capsid andhaving packaged therein an expression cassette encoding an anti-Her2 IgGantibody or a functional fragment thereof which comprises an anti-Her2heavy chain which has disrupted binding for FcRn.
 14. A method forretarding the growth of a tumor in the brain, said method comprisingadministering a composition according to claim 1 to a subject in needthereof.
 15. The method according to claim 14, wherein said compositionis administered intrathecally.
 16. The method according to claim 14,wherein said composition is administered in the absence of chemical orphysical disruption of the blood brain barrier.
 17. A method fortreating metastatic breast cancer in the brain, said method comprisingadministering a composition according to claim 1 to a subject in needthereof.
 18. The method according to claim 17, wherein said compositionis administered intrathecally.
 19. The method according to claim 17,wherein said composition is administered in the absence of chemical orphysical disruption of the blood brain barrier.
 20. An anti-neoplasticregimen comprising administering a composition according to claim 1 incombination with a biologic drug, a small molecule, anti-neoplasticagent, radiation, and/or a chemotherapeutic agent.